Neural crest cells are migratory, invasive multipotent cells that form diverse vertebrate structures, including the craniofacial skeleton. To date, most research on early neural crest development has focused on the network of transcription factors that specifies neural crest progenitors in the ectoderm. However, expression of these transcription factors does not guarantee eventual migration as a neural crest cell. Furthermore, it is not understood how the migratory machinery is deployed and coordinated to achieve neural crest cells'complex migratory behaviors. One possibility is that post-translational modifications modulate and diversify the activity of neural crest regulatory factors as cells delaminate and migrate. To investigate this possibility, previous work defined the novel protein Paladin as an antiphosphatase that is required for neural crest development. As a first step in revealing the mechanism of this requirement, two-hybrid screening identified myosin heavy chain 9 (MYH9) as a candidate phosphorylation-dependent target of Paladin. MYH9 is phosphorylated, regulates cell motility (including metastasis), and associates with non-syndromic cleft lip and palate. However, MYH9 has not been characterized in the neural crest. This application tests the hypothesis that Paladin regulates neural crest development by protecting the phosphorylation status of target proteins like MYH9 that are crucial in neural crest cells.
Aim 1 will demonstrate that MYH9 and its interaction with Paladin are relevant to neural crest cells through gene and protein expression analyses and co-immunoprecipitation studies.
Aim 2 will determine the events in neural crest development that require MYH9 as a prelude to defining the importance of MYH9 phosphorylation for these functions. Finally, Aim 3 will visualize MYH9 tyrosine phosphorylation in order to evaluate the impact of Paladin on this phosphorylation. Altogether, these studies will determine the role of MYH9 in early neural crest development and characterize MYH9 as a phosphorylated protein regulated by Paladin in the neural crest. These outcomes will create a clearer understanding of the molecular mechanisms of early neural crest development and will validate the use of Paladin to identify neural crest proteins regulated by phosphorylation. Understanding neural crest development in its normal, embryonic context will allow us to decipher the abnormal function and activation of neural crest pathways in disease.
The neural crest is a migratory stem cell population that forms the bones of the face and neurons outside the brain and spinal cord in embryos with backbones. The proposed research aims to determine the fundamental mechanisms that regulate how neural crest cells form and move. Because some metastatic cancer cells recapitulate neural crest migration, and abnormalities in neural crest development cause numerous birth defects, this information can be used to develop new therapies to target these disorders.